G2C::Proteomics

1. What is the postsynaptic density (PSD)?

The PSD is a subcellular structure of neuronal synapses, particularly of excitatory synapses. It was originally identified with electron microscopy as an electron-dense region below the postsynaptic membrane, hence its name. Proteomic studies of this structure have shown it contains several hundred different proteins including neurotransmitter receptors and many relevant molecules used to integrate and respond to synaptic input.

2. What was the experimental design?

Nine human cortical samples derived from neurosurgery were used in this study. Neuropathological examination revealed all tissue was normal. The nine fragments of tissue were pooled into three groups and postsynaptic density (hPSD) fractionation was performed for each group. hPSD fractions contain hundreds of proteins. To facilitate their detection by the mass spectrometer (MS) all samples were first separated by mass (using SDS-PAGE), proteins then fragmented into peptides, which were further separated by their hydrophobic profile (using reverse-phase liquid chromatography) and finally, injected into a the MS (LTQ FT Ultra hybrid mass spectrometer; Thermo Electron). The set of proteins identified in all three replicas was named as the consensus hPSD and was extensively analysed using bioinformatic tools.

3. What is proteomic mass spectrometry?

Mass spectrometry is a technique which measures molecular mass; if the molecules analyzed are proteins or peptides we talk about proteomic mass spectrometry. A mass spectrometer resolves the mass of a molecule by measuring the mass-to-charge ratio of its ions. Therefore ionizing proteins/peptides is essential to proteomic mass spectrometry. In a mixture of proteins of unknown composition the very accurate measure of its components mass will allow for their identification. Mass spectrometry can identify up to few thousands of proteins in complex mixtures.

A recent review of protemic methods including mass spectrometry is found in:

4. What is dN/dS?

If a nucleotide substitution in a given codon does not result in an amino acid substitution then is regarded as a synonymous or silent nucleotide substitution. If a nucleotide substitution alters a codon sequence sufficiently to alter the resulting amino acid then it is regarded as a non-synonymous substitution. Synonymous nucleotide substitutions are assumed to be neutral, because they do not alter the protein structure and therefore do not have a subsequent effect on the fitness of the organism.

For a given gene or set of genes it is possible to calculate all synonymous substitutions (represented by dS) and non-synonymous substitutions (represented by dN) that have occurred between two species considered. The ratio of dN to dS can be used as a measure of the type and degree of selection that a gene has undergone. For a given gene, if dN = dS then the gene is said to have evolved neutrally. If dN is significantly lower than dS than there is constraint against amino acid substitutions arguing for conservative, purifying selection. If dN is significantly greater than dS, then amino acid substitutions have come to prevalence in a particular lineage faster than would be expected by neutral evolution, arguing for positive Darwinian selection or adaptive evolution.

5. What is Online Mendelian Inheritance in Man (OMIM) Database?

OMIM is a database of human genes and phenotypes (http://omim.org/). It does contain information on all known mendelian disorders, focusing on the relationship between phenotype and genotype.

6. How are mammalian phenotypes identified?

Mammalian phenotypes have been derived from the database Mouse Genome Information (MGI). The phenotypic information obtained from spontaneous, induced, and genetically-engineered mutations is curated by MGI from the literature and introduced into a fixed nomenclature (or ontology) scheme which allows for comparison between mutants and bioinformatic analysis.

7. How are human phenotypes identified?

The human phenotypic data was retrieved from the Human Phenotype Ontology (HPO) database. HPO curated the phenotypic information contained in Online Mendelian Inheritance in Man (OMIM) database for monogenic diseases, and translated into a fix nomenclature (or ontology). OMIM phenotypic information is derived by searching the literature in which diseases are described and cases are reported. The nomenclature created by HPO does allow for comparison of phenotypes between genes or vice versa.